Molecular dissecting and targeting YAP1 mediated cancer stemness and immune suppression in advanced gastric adenocarcinoma

项目摘要 本研究的主要目的是阐明YAP 1介导的转移小生境的机制， 免疫抑制治疗胃癌腹膜癌病 发现。GAC是美国和全球的主要健康负担。PC常见，影响约45%的GAC 患者GAC合并PC患者生存期短，治疗无效。分子理解 PC是有限的。为了应对这一尚未解决的临床挑战，我们建立了PC银行基础设施，旨在 利用这些患者来源的标本来发现和验证我们的新靶点。我们的初步RNAseq PC标本的分析显示了独特的免疫抑制分子的富集，包括TIM 3和 其配体为半乳糖凝集素9（Galectin-9）、TGF-β和VISTA，而PD-1/PDL-1和CTLA-4表达较少。YAP 1具有 与人类发育、谱系可塑性有关，并在许多肿瘤类型中上调。但其 在介导PC转移和肿瘤微环境（TME）中的免疫抑制中的作用尚不清楚。 我们的初步数据表明，YAP 1在原发性和转移性GAC肿瘤细胞中高表达 患者，并与生存率低显著相关。基因敲除（KO）YAP 1显著降低 小鼠的癌干特性、肿瘤形成和PC。此外，在肿瘤细胞中YAP 1的消耗一致地 增加来自GAC的CD 3和CD 8 T细胞应答。通过PC样品的单细胞RNAseq（scRNAseq） 通过免疫荧光染色验证，我们注意到YAP 1、TIM 3配体Gal-9和DKK 1高度表达， 在PC的肿瘤细胞中表达;而TIM 3在免疫细胞中富集。来自YAP 1high和YAP 1 KO的RNAseq 患者来源的肿瘤细胞显示，Gal-9和DKK 1在YAP 1耗尽后显著降低， 这些因素与患者的生存率低有关。我们假设YAP 1high PC细胞是 转移起始细胞通过赋予肿瘤细胞癌症干细胞属性来协调小生境 并通过激活TIM 3/Gal-9轴和增加DKK 1旁分泌而促进肿瘤免疫抑制 在PC TME因此，同时靶向Hippo/YAP 1和免疫检查点（TIM 3/Gal-9）可能是一种有效的免疫抑制剂。 增强战略。为了验证我们的假设，我们提出了三个具体目标：目标1。确定函数 使用新的干细胞克隆技术研究YAP 1与肿瘤细胞中PC干细胞和转移的相关性， 体内PDX/PDO模型。目标2.研究YAP 1介导免疫抑制的机制 在PC的TME中。目标3：使用以下方法阐明单独抑制YAP 1或与TIM 3抑制组合抑制YAP 1的功效 PDO模型、KP-Luc 2同基因小鼠模型、GEMM和正在进行的YAP 1临床试验。通过利用患者- 衍生的PC细胞，我们将揭示YAP 1介导的PC功能的重要性，并阐明其机制， 其中YAP 1介导的免疫抑制用于新的治疗策略。在完成这项研究后，我们 将有一个强有力的理由，一个新的组合，可以克服我们在经验的缺点， 今天的诊所

Project Abstract The main objectives of this proposal are to elucidate the mechanisms of YAP1-mediated metastatic niche and immunosuppression in gastric adenocarcinoma (GAC) with peritoneal carcinomatosis (PC) for novel therapeutic discoveries. GAC is a major health burden in the US and worldwide. PC is common affecting ~45% of GAC patients. GAC patients with PC have short survival and treatments are ineffective. Molecular understanding for PC is limited. To address this unmet clinical challenge, we have established PC banking infrastructure aiming to utilize these patient-derived specimens to discover and validate our novel targets. Our preliminary RNAseq profiling of PC specimens revealed an enrichment of unique immune suppressive molecules, including TIM3 and it’s ligand Galectin-9 (Gal-9), TGF-β and VISTA, but less expression of PD-1/PDL-1 and CTLA-4. YAP1 has been implicated in human development, lineage plasticity, and upregulated in many tumor types. However, its role in mediating PC metastases and immune suppression in tumor microenvironment (TME) remain unclear. Our preliminary data suggest that YAP1 is highly expressed in primary and metastatic tumor cells of GAC patients and is significantly associated with poor survival. Genetic knockout (KO) YAP1 significantly decreased cancer stemness traits, tumor formation and PC in mice. Further, depletion of YAP1 in tumor cells consistently increased CD3 and CD8 T cell responses from GAC. Through a single cell RNAseq (scRNAseq) of PC samples and validation using immunofluorescent staining, we noticed that YAP1, TIM3 ligand Gal-9 and DKK1 are highly expressed in tumor cells of PC; while TIM3 is enriched in immune cells. RNAseq from YAP1high and YAP1 KO patient-derived tumor cells revealed that Gal-9 and DKK1 were significantly decreased upon depletion of YAP1 and these factors are associated with poor survival of patients. We hypothesize that YAP1high PC cells are metastasis-initiating cells that orchestrate a niche by conferring cancer stemness attributes to the tumor cells and promote tumor immunosuppression through activating TIM3/Gal-9 axis and increasing paracrine of DKK1 in PC TME. Therefore, simultaneously targeting Hippo/YAP1 and immune checkpoint (TIM3/Gal-9) could be an enhanced strategy. To test our hypothesis, we propose three Specific Aims: Aim 1. Determine the functional relevance of YAP1 in PC stemness and metastases in tumor cells using novel stem cell cloning technology and PDX/PDO models in vivo. Aim 2. To investigate the mechanisms whereby YAP1 mediates immunosuppression in TME of PC. Aim 3. Elucidating efficacy of inhibition of YAP1 alone or in combination with TIM3 inhibition using the PDO models, KP-Luc2 syngeneic mouse model, GEMM, and ongoing YAP1 clinical trial. By utilizing patient- derived PC cells, we will uncover functional importance of YAP1-mediated PC and elucidate the mechanisms by which YAP1 mediated immunosuppression for novel therapeutic strategies. Upon completion of this study, we will have a strong rationale for a novel combination that could overcome the shortcomings we experience in the clinics today.